Abstract Zika virus (ZIKV) is a mosquito borne flavivirus that recently emerged in the Americas. ZIKV infection is associated with severe disease outcomes, including Guillain-Barr syndrome and microcephaly, a devastating congenital disorder where fetal brain development is impaired. Despite ZIKV?s status as a significant emerging disease threat, there are currently no approved vaccines or therapies for ZIKV infection. Thus a better understanding of ZIKV replication is needed to guide both vaccine and therapy design. While relatively little is known about ZIKV virulence determinants, a growing body of evidence suggests that RNA structural elements in RNA viral genomes play a major role in virus replication and pathogenesis. The full complement of RNA secondary structures within the ZIKV genome is not known, nor is it understood how these structures affect replication. We therefore used selective 2?-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) to define the RNA structural landscape of the entire ZIKV genome of a clinical isolate from the ongoing ZIKV epidemic in the Americas. This analysis provided the first comprehensive view of RNA structure in the ZIKV genome. We identified RNA secondary structures distributed throughout the genome, including 19 regions with exceptionally high structural stability. These highly stable structures include known structures within the ZIKV 5? UTR and 3? UTR, thus validating the use of SHAPE-MAP to identify ZIKV RNA structures. The additional highly stable RNA structures are present across the coding region of the ZIKV genome and have not been characterized. These results demonstrate that the ZIKV genome is highly structured, and we hypothesize that the novel RNA structures may play important roles in multiple aspects of the ZIKV lifecycle. The fact that known flavivirus RNA structures are highly conserved suggests that some of the novel ZIKV structures are also conserved across flaviruses and may function as virulence determinants. Therefore, in Aim 1 we test whether novel, highly stable RNA structures in the ZIKV genome are important for ZIKV replication and pathogenesis. To determine the level of RNA structural conservation between ZIKV and other flaviviruses, in Aim 2 we use SHAPE-MaP to comprehensively define RNA structure in the entire genome of several other medically important flaviviruses (Yellow Fever virus, West Nile virus, and Powassan virus). These studies will significantly advance our understanding of how RNA structure impacts ZIKV biology, and provide new insights into whether highly conserved RNA structures broadly contribute to flavivirus replication and pathogenesis.